Generation of transgenic mice by transgene-mediated rescue of spermatogenesis

ABSTRACT

The present invention provides transgenic mice and nucleic acid constructs and methods for creating them. The transgenic mice are generated preferably by electroporation-mediated transfection of a nucleic acid construct into the testes of a sterile spermatogenesis-deficient male mouse. The construct includes DNA encoding a spermatogenesis rescue factor resulting in rescue of spermatogenesis while contemporaneously transferring a nucleic acid of interest for expression in the transgenic mouse so that progeny sired by the transgenic mouse also harbor the transgene. Certain embodiments of the invention also utilize (a) the salmonid derived Sleeping Beauty transposition system to augment genomic integration of the transgene and/or (b) the Cre/loxP recombination system for excision from sired progeny of the DNA responsible for spermatogenesis rescue.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention in the field of molecular and developmental biologyrelates to a novel method for generating transgenic mammals, geneticconstructs useful therein, and novel genetically modified mammals.

2. Description of the Background Art

In the most widely used method for producing transgenic animals, DNA ismicroinjected into the pronuclei of one-cell stage embryos (Gordern etal 1980. Proc. Natl. Acad. Sci. USA 77:7380-7384. The resulting animalsare propagated, transmitting the transgene in the germline (Brinster etal. (1981) Cell 27:223-231; Costantini and Lacy(1981), Nature 294:92-94;Gorden and Ruddle (1991) Science 214:1244-1246; E. Wagner et al. (1981)Proc. Natl. Acad. Sci. USA 78:5016-5020; T. Wagner et al.(1981) Proc.Natl. Acad. Sci. USA 78:6376-6380). Wagner and Hoppe, U.S. Pat. No.4,873,191, disclose genetic transformation of a zygote (typicallypronuclei of a male zygote) by microinjection of exogenous geneticmaterial into the nucleus which ultimately forms at least a part of thenucleus of the zygote. The zygote is then allowed to undergodifferentiation and development into the organism. The genotype of thezygote, and the resulting organism, includes the gene(s) present of theexogenous genetic material which is phenotypically expressed.

In spite of its widespread use, the method described above is tedious.Generally, three to four hundred eggs are needed for microinjection,requiring collection from at least ten superovulated female mice whoreceive hormone injections one day before egg collection after priminghormonally (e.g., with pregnant mare serum) two days before mating.These females are mated to males, and fertilized eggs are collected thefollowing morning. DNA constructs are injected into male pronuclei withthe aid of a specialized microscope after which the eggs are surgicallyimplanted into the oviducts of pseudopregnant females that had beenmated to vasectomized males the day before ((Hogan et al. (1994)Manipulating the Mouse Embryo: A Laboratory Manual, 2^(nd) Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 218-252).

Other methods of introducing exogenous DNA into embryos rely on viralinfection. Three major approaches have been employed. One group ofmethods relies on infection of early mouse embryos by co-culture withretrovirus-producing cells (Hogan et al., supra, pp. 251-252).Alternative methods use infection of either pre- or post-implantationmouse embryos with wild-type or recombinant retroviruses (Jaenisch etal. (1976) Proc. Natl. Acad. Sci. USA 73: 1260-1264; Jaenisch et al.(1981) Cell 24: 519-529; Stuhlmann et al. (1984) Proc. Natl. Acad. Sci.USA 81: 7151-7159; Jahner et al. (1985) Proc. Natl. Acad. Sci. USA 82:6927-6931; Van der Putten, et al. (1985) Proc. Natl. Acad Sci. USA 82:6148-6152; Stewart, et al.(1987) EMBO J. 6: 383-388). In yet anotherapproach based on viral infection, the retrovirus, or virus-producingcells, are injected into the blastocele (Jahner et al. (1982) Nature298: 623-628). The foregoing methods have not gained wide acceptancebecause they all require laborious virus production and result inmosaicism.

Direct gene transfection into the adult germline has rarely beenattempted due to difficulties in manipulating germ cells; limitedsuccess was reported with retrovirus-mediated gene delivery into malegerm cells (Nagano (2000) FEBS Letters 475:7-10). Limitations of thisapproach arise from the lack of reliable culture methods that wouldpermit long-term proliferation of germ cells, difficulties in geneintegration, and unproven methods for selection in culture (Watanabe etal. (1997) Exp. Cell Res. 230: 76-83).

If successful, however, germline transfection would provide variousimportant advantages over conventional methods. First, such directmodification of the germline would be the most efficient transgenesismethod in some species by avoiding the tedious conventional proceduresof embryo collection and transfer. Second, it would enable the study ofgene function in germ cell development and gametogenesis immediatelyafter transfection, even in cases when the transgene is known to causeembryonic lethality which eliminates production of fully transgenicoffspring (Huang et al., supra). Finally, such direct methods may be theonly ones that permit study of the integration of foreign vectors intochromosomes around the time of initiation of meiosis; the only periodwhen endogenous homologous recombination occurs frequently along thechromosomes.

Chang and colleagues generated transgenic mice by lipofection of atransgene construct into mouse testes (Chang et al. (1999) J. Reprod.Dev. 45: 3742). This method, however, proved unreliable and inefficient.Furthermore, the transgenic mice failed to pass the transgene to siredprogeny. See also Sato et al. (1999) Transgenics 3: 11-22.

The application of electroporation to mouse testes has so far met withlimited success. Studies by Yamazaki and colleagues showed expression ofmarker genes two months after transfection in clusters of spermatogeniccells. However, very few spermatogenic cells were shown to carry, letalone express, the transgene. The study also failed to show expressionof marker genes in mature spermatozoa (Yamazaki et al. (1998) Biol.Reprod. 59: 1439-1444), a prerequisite for their passage to progeny. Thesame investigators were unable to produce transgenic progeny mice siredby initial transfectants. In the latter study (Yamazaki et al. (2000) J.Exp. Zool. 286:212-218), the investigators successfully transferred agene encoding green fluorescent protein (GFP), a well-known marker, invivo to mouse spermatogenic cells. GFP was expressed in transfectedtestes not only in subsurface regions but also in the inner region ofthe testes indicating that the gene was transferred throughout thetestis. GFP expression, however, remained transient. More importantly,there was no evidence of transgene transmission to offspring aftermating. The total number of male germline cells with integratedtransgene was low by histological analysis. The likely explanation wasthat the majority of sperm remained non-transgenic after electroporationand out-competed those sperm that were transgenic in the fertilizationprocess.

The present invention is intended to solve these problems.

Spermatoenesis

Spermatogenesis is the three-step process by which stem cells developinto mature spermatozoa: spermatocytogenesis (mitosis), meiosis, andspermiogenesis. In the spermatocytogenesis stage, stem cells (Type Aspermatogonia) divide mitotically to produce Type B spermatogonia thatbegin to differentiate by meiosis. During meiosis, cells in prophase ofthe first meiotic division form the primary spermatocytes, which arecharacterized by highly condensed chromosomes and intermediate positionin the seminiferous epithelium. Primary spermatocytes proceed throughthe first meiotic division and become haploid secondary spermatocytes.The products of the second meiotic division are spermatids.

During spermiogenesis spherical spermatids metamorphose into elongatedspermatozoa, the acrosome and the flagellar apparatus form, and mostexcess cytoplasm (the residual body) is separated and left in theSertoli cell. Spermatozoa are released into the lumen of theseminiferous tubule. At all stages of differentiation, the spermatogeniccells are in close contact with Sertoli cells, which are thought toprovide them with structural and metabolic support. Of particularinterest to the present invention are the genetic factors that controlspermatogenesis.

SUMMARY OF THE INVENTION

The present invention provides a novel method of transgenesis andgeneration of transgenic mice that simplifies the laborious andtime-consuming conventional pronucleus injection approach.

In a preferred embodiment of this method, a sterile male mouse deficientin spermatogenesis is transfected, preferably by electroporation, with a“transgenic construct” (which term is used to indicate a constructcarrying a nucleic acid sequence to be incorporated into a hostmammalian genome as a “transgene”). The transgenic construct alsocomprises a “spermatogenesis rescue cassette” (“SRC”).

As used herein, “spermatogenesis rescue cassette” (“SRC”) means anucleic acid construct which, when expressed in a host deficient inspermatogenesis, overcomes that deficiency and restores normal ornear-normal sperm production. A preferred SRC includes a nucleic acidsequence encoding a functional “spermatogenesis essential factor” (or“SEF”) which is a protein that is essential for spermatogenesis and,therefore, for normal or near normal male fertility. Known deleteriousmutations in SEF genes lead to arrest of sperm production and sterility(Pittman, D L, et al. (1998) Mol. Cell. 1: 697-705).

The SEF in the preferred SRC is operably linked to a promoter that isoperative in a mammal, preferably a mouse. A preferred promoter is thatof the histone H1t gene (described by Bartell, J G et al., 1996, J.Biol. Chem. 271:4046-4054, for rat). The H1t gene is expressedexclusively in pachytene spermatocytes in mice (Drabent, B. et al.,1998, Cell Tissue Res 291:127-132).

In one embodiment, two loxP recombination sites flank the SRC in thetransgenic construct. The loxP sites direct Cre recombinase-mediatedexcision of the SRC in transgenic progeny while the nucleic acid ofinterest remains incorporated in the genome.

Another embodiment utilizes the “Sleeping Beauty” (“SB”) Transpositionsystem to augment integration of the gene of interest into the recipientgenome. In one embodiment, the transgenic construct comprises an SRCoptionally flanked by loxP recombination sites and a gene of interest,all of which is flanked by the nucleic acids sequence of the invertedrepeated regions recognized by SB transposase. In this embodiment,sterile male mice deficient in spermatogenesis are co-transfected with atransgenic construct and a plasmid, preferably pCMV-SB, containing theSB transposase encoding gene. In another embodiment, such mice aretransfected with the transgenic construct and mated to female micetransgenic for DNA encoding SB transposase (and Cre recombinase if theCre/loxP recombination system is also used). In yet another embodimentDNA encoding the SB transposase is part of the construct comprising thegene of interest.

Specifically, the present invention is directed to a transgenic mousewhose germline cells comprise a transgene which includes: (a) a nucleicacid of interest, expressible in cells of the transgenic mouse,optionally operatively linked to a first promoter; and (b) an SRCcomprising a nucleic acid encoding a SEF operatively linked to a secondpromoter, which SRC is optionally flanked at its 5′ and 3′ end by a loxPrecombination site. In another embodiment, the transgenic mouse's cellscomprise (a) a transgenic nucleic acid of interest, expressible in cellsof the transgenic mouse, operatively linked to a first promoter; and (b)a single loxP recombination site either upstream or downstream from thenucleic acid. Preferred SEF's include Dmc1, Prp8 and Ccna1 (whichencodes Cyclin A1).

In the above transgenic mouse the first promoter can, for example, bethe hCMV promoter or a hEF promoter or any promoter operative in mice.The second promoter can also be any promoter operative in mice and ispreferably the H1t promoter or a promoter natively linked to the SEFgene.

In another embodiment of the above transgenic mouse, the transgenicconstruct is flanked at its 5′ and 3′ ends by a nucleic acid sequenceencoding SB transposon inverted repeats or partially functional mutantsthereof. The nucleic acid of interest comprised within the transgenicconstruct flanked by the SB transposon, preferably becomes integrated inthe genome in more than one location.

Also provided is a nucleic construct for introducing a transgene into amammal comprising: (a) a nucleic acid of interest which is optionallyoperatively linked to a first promoter, (b) a SRC comprising a nucleicacid encoding a SEF operatively linked to a second promoter. In thisconstruct the SRC may be flanked at its 5′ and 3′ end by a loxPrecombination site. Preferred SEF's in the above construct include Dmc1,Prp8 and Ccna1. In this construct, the first or second promoter is anhCMV promoter or an hEF promoter. Another preferred second promoter isthe H1t promoter, a promoter that is natively linked to the SEF gene.The nucleic acid of interest may be flanked at its 5′ and 3′ ends by anucleic acid sequence encoding the SB transposon inverted repeats orflanked by a partially functional mutant thereof.

The above construct containing the SB inverted repeats may furthercomprise a nucleic acid sequence encoding a functional transposase(linked to a promoter) that recognizes the inverted repeats.

The present invention is also directed to a method of producing atransgenic mouse that expresses as a nucleic acid of interest,comprising the steps of:

-   (a) transfecting germ cells of a sterile spermatogenesis-deficient    immature male mouse with the above which incorporates both the SRC    and the nucleic acid of interest into the germ cells, thereby    generating a transfectant mouse;-   (b) permitting expression of the SEF in the transfectant mouse,    thereby rescuing spermatogenesis;-   (c) waiting a sufficient period for the transfectant mouse to mature    sexually;-   (d) mating the transfectant mouse to a female mouse to create F₁    progeny mice; and-   (e) mating a resulting F₁ progeny mouse to a fertile mouse,    thereby producing the transgenic mouse whose genome includes the    nucleic acid of interest.

Another embodiment of this method comprises:

-   (a) transfecting germ cells of a sterile spermatogenesis-deficient    immature male mouse with the above which incorporates both the SRC    and the nucleic acid of interest into the germ cells, thereby    generating a transfectant mouse;-   (b) permitting expression of the SEF in the transfectant mouse,    thereby rescuing spermatogenesis;-   (c) waiting a sufficient period for the transfectant mouse to mature    sexually,-   (d) mating the transfectant mouse to a female mouse that is    transgenic for DNA encoding Cre recombinase to create F₁ progeny    mice, wherein, when the construct includes the lox P sites, the    recombinase mediates excision of the SRC from the DNA of the F₁    progeny mice; and-   (e) mating a resulting F₁ progeny mouse to a fertile mouse,    thereby producing the transgenic mouse whose genome includes the    transgenic nucleic acid of interest but not the SRC.

In one embodiment of the above method (i) the construct includes theloxP sites, and (ii) the female mouse of step (d) is transgenic for DNAencoding Cre recombinase, so that in the F₁ progeny mice, therecombinase mediates excision of the SRC from the DNA, thereby producinga transgenic mouse whose genome includes the transgenic nucleic acid ofinterest but not the SRC.

In the above method, transfecting is preferably accomplished byelectroporation but may be accomplished by other means such as lipidmediated transfection, nucleic acid-coated microprojectile bombardment.

In a preferred embodiment of the above method, (a) in the construct, thenucleic acid of interest is flanked at its 5′ and 3′ ends by (i) nucleicacid sequences encoding SB transposon inverted repeats or (ii) partiallyfunctional mutants of the repeat-encoding sequences, and (b) the femalemouse is also transgenic for DNA encoding (i) a transposase thatrecognizes the inverted repeats to which is linked a promoter. Themethod further comprises, after the mating step (d) above, the step ofinducing transposase mediated transposition of the nucleic acid sequenceof interest which thereby integrates in one or more sites of the genomeof the F₁ progeny mouse.

Alternatively, a transgenic construct comprising the SB inverted repeatscan be co-transfected along with a plasmid, preferably pCMV-SB,comprising DNA encoding the SB transposase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation for generating transgenic miceusing the present invention. The testes of a sterile, Dmc1^(−/−) malemouse are transfected with the transgenic construct shown in FIG. 2. Themale transfected mouse matures and is mated to a female transgenic forCre recombinase to produce F₁ progeny. The spermatogenesis rescuecassette (SRC) within the transgenic construct is excised in the F₁progeny by way of Cre/loxP recombination. These F₁ progeny are thenmated to wild type mice to yield progeny in which the Cre recombinasegene segregates out and the transgene-derived nucleic acid of interestis incorporated in the genome.

FIG. 2 shows an embodiment of the transgenic construct used in thisinvention. This transgenic construct comprises a SRC which includes anucleic acid encoding a spermatogenesis essential factor (“SEF”) such asDmc1, Prp8, or Ccna1 under the control of a promoter operative inmammalian, preferably murine, cells. The SRC is flanked by loxP sites.The transgenic construct also comprises a nucleic acid of interest(shown as “Gene X”) under the control of a second promoter operative inmammalian, preferably murine, cells.

FIG. 3 shows another embodiment of the transgenic construct used in thisinvention. This construct is similar to that depicted in FIG. 2 andfurther comprises 5′ and 3′ flanking nucleic acid sequences encoding theinverted repeats of the SB transposon.

FIG. 4 shows a schematic representation for generating transgenic micein a similar way as shown in FIG. 1 using the transgenic constructdepicted in FIG. 3.

The foregoing figures are provided for illustration purposes and in noway limit the scope of the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The key concept underlying this invention is the use of a male mousethat has been rendered sterile due to a mutation or mutations in a geneor genes encoding a “spermatogenesis essential factor” such as Dmc1,Prp8, or Ccna1. A transgenic construct such as that shown in FIG. 2containing a wild type version of the mutant SEF gene is introduced intomouse testicular germ cells preferably by electroporation or othermeans. The expression of the wild type gene in the mutant germ cellsrestores normal spermatogenesis and promotes development of functionalsperm. Since the mutant germ cells cannot produce functional spermwithout expression of the SEF transgene, functional sperm that have beenrescued by the transgenic construct also harbor the nucleic acid ofinterest that was part of that construct. When these transgenic malemice are later mated to normal females, their progeny will inherit thetransgene (FIG. 1).

Transgenic Construct

The transgenic construct, preferably linear, consists of two componentsas illustrated in FIGS. 2 and 3. The first component is thespermatogenesis rescue cassette, the SRC, having a wild type nucleicacid, preferably DNA, encoding an SEF used to rescue the mutantphenotype. Examples of the preferred SEF's are Dmc1, Prp8, and Ccna1.

The expression of this nucleic acid is preferably under the control of agerm cell-specific promoter, preferably, the H1t gene promoter (Kremer EJ et al., 1992, Gene 110:167-173), although other, ubiquitous promoterscan also be used.

Gene H1t encodes a testis-specific variant of the H1 histone familyexpressed in pachytene spermatocytes during the meiotic phase ofspermatogenesis. In the initial description of this promoter, expressionof the minimal promoter (174 nucleotides (nt) upstream from thetranscription start point) was enhanced by sequences extending tont-693, but was reduced in constructs with kb of upstream sequence. TheH1t promoter is modulated both positively and negatively by distantupstream sequences in the native Ht1 gene structure.

In another embodiment, the promoter is an endogenous promoter of thegene encoding the SEF.

If desired, the SRC is flanked by loxP sites that will later facilitateexcision of the rescue cassette, although such excision is optional. Infact, use of the CRE/loxP system as described herein is optional in thepresent methods.

The second component of the transgenic construct is the nucleic acidbeing inserted into the mouse's genome, i.e., the nucleic acid ofinterest, which is operatively linked to a separate promoter that isoperative in mammalian, preferably murine, cells.

The promoter can be any one that drives expression of the nucleic acidof interest in mouse (male germline) cells. The promoter may be viral(e.g. hCMV promoter) or eukaryotic, preferably mammalian. Suitablepromoters are inducible or repressible or, more preferably,constitutive. Preferred eukaryotic/viral promoters include the promoterof the mouse metallothionein I gene (Hamer, D., et al., J. Mol. Appl.Gen. 1:273-288 (1982)); the TK promoter of Herpes virus (McKnight, S.,Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, C., et al.,Nature 290:304-310 (1981)); and the yeast gal4 gene promoter (Johnston,S. A., et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver,P. A., et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)). Auseful non-mammalian eukaryotic promoter is carp β-actin gene promoter(FV) (Ivics et al., Cell (1997) 91:501-510). Examples of vectors(plasmid or retrovirus) are disclosed in (Roy-Burman et al., U.S. Pat.No. 5,112,767). All of the above listed references are incorporated byreference in their entirety.

An example of a constitutive promoter is the viral promoter MSV-LTR,which is efficient and active in a variety of cell types, and, incontrast to most other promoters, has the same enhancing activity inarrested and growing cells. Other preferred viral promoters include thatpresent in the hCMV-LTR (from cytomegalovirus) (Bashart, M. et al., Cell41:521 (1985)) or in the RSV-LTR (from Rous sarcoma virus) (Gorman, C.M., Proc. Natl. Acad. Sci. USA 79:6777 (1982). Another preferredpromoter is the human elongation factor 1α (“hEF”) promoter (Kim, D W etal., (1990) Gene 91:217-223).

Delivery of Transgenic Construct into Mouse Spermatogenic Cells

In a preferred embodiment, young male mice at postnatal day 14 aresubjected to general anesthesia (e.g., with 2.5% avertin at a dose of0.015-0.017 ml/g of body weight). Testes are pulled out with bluntforceps and allowed to rest on the fat pad. The transgenic construct inthe form of a nucleic acid molecule preferably in a volume of 10-50 μl,is injected into the efferent ductules between testes and epididymis. Inthe electroporation embodiment described in more detail below, afterinjection the testes are held between a pair of electrodes(Tweezertrode, BTX) and 8 square 50 msec pulses at 10-50 V, are applied(BTX ECM 830 electroporator) to effect electroporation and uptake of theDNA. The testes are then reinserted into the body wall. The incision isclosed with two to three stitches both for the body wall and the skin.

Electroporation

Electroporation, also known as electropermeabilization, is popularlyused for transfection of cell suspensions. Forcep electrodes are usedfor in vivo electroporation aimed at various organs and tissue types(Muramatsu et al. (1998) J. Mol. Med. 1: 55-62). Electroporation entailsexposing cells to short intense electric field pulses thereby inducing atransmembrane potential. The applied field induces temporary structuralchanges in the cell membrane, creating pathways from the extracellularspace into the cell interior (Jaroszeski et al. (2000) Meth. Mol. Med.37:173-186).

Currently, transmembrane induction voltages between 0.5 and 1 V minimumpotential are used for most mammalian cell types. Induction potentialsabove this threshold can cause irreversible membrane damage and celldeath (Teissié et al. (1992), In: Charge and Field Effects in Biosystems(Allen, M. J et al. eds.) Birkhauser, Boston, 3: 285-301); Teissié etal. (1982) Science 216:537-538).

Apart from the extent of membrane permeabilization, determined primarilythrough pulse duration and voltage, other factors control the intake ofexogenous DNA by the cell. Most electroporation-mediated exogenous DNAtransport is through electrophoresis rather than solely by membranepermeability (Klenchin et al. (1991) Biophys. J. 60:804-811, Wolf etal., Biophys. J. 66:524-531). Adding DNA immediately after the pulseusually results in lower transfection efficiency than when the DNA isadded prior to the pulse. Shielding the charge of DNA by cations alsoreduces transfection efficiency (Anderson et al. (1989) 180: 269-275).

Most of the DNA entering the cell does so during the pulses by way ofthe electric field created across the membrane. As such, thetransfection efficiency is proportional to the integral of the extent ofmembrane permeabilization with respect to the pulse time. For simplerectangular pulses or exponentially decaying pulses, the time integralis the pulse length T, which is usually much greater than the membranerelaxation time. If the permeabilized area of the cell is limited topolar regions, as is normally the case, the extent of membranepermeabilization (in terms of the number, density, and size ofelectropores) is approximately proportional to E-E_(b), where E_(b) isthe pulse field strength needed to produce the membrane breakdownvoltage V_(b) (Hibino et al. (1991) Biophys. J 59: 209-220). Thus thetransfection efficiency is roughly proportional to (E-E_(b))T (Hui etal. (2000) Meth. Mol. Med. 37: 157-171).

Nucleic Acid-Coated Microprojectile Bombardment

This method involves propulsion of nucleic acid-coated microprojectiles(preferably DNA) into target cells (Sanford et al. (1988) Particle Sci.Technol. 5: 27-37). Gold particles are particularly preferred.

A commercially available device (Biolistic PDS-1000; from Du Pont) usesa gunpowder discharge to impart momentum to coated projectiles. Whenperformed in vitro, target cells are placed in a vacuum chamber duringbombardment to minimize air impedance of particle flight.

Williams et al., (2000) Proc. Natl. Acad. Sci. USA 88:2726-2730,disclose a device for microprojectile bombardment to introduce andexpress exogenous nucleic acids directly in intact tissue of the livingmouse. This device uses a helium discharge system, a disc macrocarrierfor microprojectiles, and is configured to be hand-held. The use ofhelium gas permits more precise regulation of particle velocity and isconfigured such that the helium discharge impelling the microprojectilesis deflected away from the tissue, minimizing damage from the resultingshock wave. The helium discharge drives the macrocarrier through a 0.8cm flight path to a stopping screen that arrests the macrocarrier discbut is permeable to nucleic acid coated microprojectiles which arepermitted to strike the target tissue. Tissue becomes bombarded withcoated gold particles (available from Alfa, Ward Hill, Mass.) having arange diameter between 1 and 3 μm or between 2 to 5 μm when tungstenparticles (Sylvania) are used.

Microparticles are coated with nucleic acid by sequentially mixing goldor tungsten in an aqueous slurry comprising nucleic acid (about 1mg/mL), CaCl₂ (2.5 M) and free-base spermidine (1 M). After 10 or sominutes of incubation, the microprojectiles are pelleted and thesupernatant removed. The pellet is washed once with 70% ethanol,centrifuged, and resuspended in anhydrous ethanol. The nucleic acidcoated microprojectiles are spread over the macrocarrier discs andallowed to dry in a dessicator before firing. The exact specificationsof the device and its use are detailed in Williams, supra, which ishereby incorporated by reference.

A person having ordinary skill in the art will recognize that otherapproaches may be employed to introduce the transgenic construct intothe germline such as viral infection or lipofection.

Lipid Mediated Transfection (Lipofection)

Some of the first work on liposome delivery of endogenous materials tocells occurred some twenty years ago. Foreign nucleic acids wereintroduced into cells using positively charged lipids. (Martin et al.,(1976) J. Cell Biol. 70: 515-526, Magee et al., (1976) Biochim. Biophys.Acta 451: 610-618, and Straub et al., (1974) Infect. Immun. 10:783-792).

Of the many methods used to facilitate entry of DNA into eukaryoticcells, cationic liposomes are among the most efficacious and have foundextensive use as DNA carriers in transfection experiments. (Thierry etal., Gene Regulation: Biology of Antisense RNA and DNA, p. 147 (Ericksonand Izant, Eds., Raven Press, New York, 1992).

Senior et al., (1991) Biochim. Biophys. Acta 1070:173, suggested thatincorporation of cationic lipids in liposomes is advantageous because itincreases the amount of negatively charged molecules that can beassociated with the liposome. In their study of the interaction betweenpositively charged liposomes and blood, they concluded that harmfulside-effects associated with macroscopic liposome-plasma aggregation canbe avoided by limiting the dosage.

U.S. Pat. Nos. 5,695,780, 5,688,958, 5,686,620, 5,661,018, 5,651,981,herein incorporated by reference in their entirety, further elaboratethe types of lipids useful in lipofection vectors and methodology usedin the lipofection of nucleic acids into eukaryotic cells.

Spermatogenesis Essential Factors (SEF)

By combining transfection of mouse germ cells in the testes with anexogenous nucleic acid molecule (whether by electroporation, bombardmentor other means), followed by integration, expression as a proteinproduct in the germline, and passage of the incorporated nucleic acid tosubsequent progeny, the present invention overcomes a limitation in theprior art (see Yamazaki et al., 2000, supra). A particular advantage ofthis invention over the art is the exploitation of spermatogenesis as aselection factor for enriching the stock of transgenic sperm availablefor fertilization.

The present invention exploits the genetic factors that controlspermatogenesis. One such SEF is Dmc1, a meiosis specific gene firstdiscovered in yeast that encodes a homologue of bacterial RecA and isimplicated in recombination (Roca et al. (1990) Crit. Rev. Biochem. Mol.Biol. 25: 415-456). Yeast Dmc1 mutants and mutants of its murinehomologue are defective in crossing over and synaptonemal complexformation and undergo arrest in late prophase meiosis I (Pittman et al.,supra). Mammalian Dmc1 homologues have been isolated from mouse andhuman cDNA libraries (Sato et al. (1995) DNA Res. 2: 147-150); Habu etal. (1996) Nucleic Acid Res. 24: 470-477).

Both mouse and human Dmc1 genes encode a 340 amino acid proteincontaining the two nucleotide binding motifs (GEFRTGKT [SEQ ID NO: 1}and LLIID [SEQ ID NO: 2]) important for binding single anddouble-stranded DNA (Ogawa (1993) Cold Spring Harb. Symp. Quant. Biol.58: 553-565). Transcription of mouse Dmc1 is restricted to testes andovary, consistent with meiosis-specific expression in yeast

-   -   Dmc1 deficient mouse mutants show normal viability. However,        crosses of Dmc1^(−/−) males to females failed to yield any        births.

Furthermore, mouse Dmc1 mutants lack any mature spermatozoa. In normalmice, spermatogonial stem cells are found at the periphery of theseminiferous tubules. The seminiferous tubules from homozygousDmc1^(−/−) mutants have arrested gamete development at the spermatocytestage. Indeed, Dmc1^(−/−) testes are completely deficient in postmeoiticcells.

Another SEF that can be exploited in the present invention is thesplicing factor Prp8. During mouse embryogenesis, Prp8 is expressedintensely at day 9.5 of gestation and its expression decreasesprogressively during embryogenesis. In adult mice, Prp8 is expressedstrongly in the testes and moderately in the ovary. In situhybridization analysis revealed that Prp8 is preferentially expressed inthe outer cell layer in the testes (spermatogonia, primaryspermatocytes, and in granulosa cells in the ovary). Unlike its yeastcounterpart, which is essentially a U5 small nuclear RNP particle,vertebrate Prp8 has acquired an additional role in reproduction andspermatogenesis (Takahashi et al. (2001) J. Biochem. 129: 599-606).

Another SEF useful in the present invention is Cyclin A1 encoded byCcna1. The mammalian A-type cyclin family consists of two members,cyclin A1 and A2. In mice, mutations in cyclin A2 are lethal whilemutations in cyclin A1 produce viable but spermatogenesis-deficientsterile male mice. (Liu et al. (1998) Nature Genetics 20: 377-380). InCcna1^(−/−) mice, approximately 10% of tubules contain spermatocytesundergoing apoptotic cell death, and the average testis weight is about61% of that in wild-type mice. Moreover, the mutants failed to produceoffspring when mated to normal females. Ccna1 is thus essential forspermatogenesis in early stage passage into the first meiotic divisionin male mice.

Cre/loxP Recombination System

One embodiment of this invention utilizes the Cre/loxP recombinationsystem (see, e.g., U.S. Pat. No. 4,959,317) for excising thespermatogenesis rescue cassette from transgenic progeny. Cre recombinaseof the P1 bacteriophage belongs to an integrase family of site-specificrecombinases that is expressed in mammalian and other eukaryotic celltypes (Saur et al. (1988) Proc. Natl. Acad. Sci. USA 85:5166-5170,(1989) Nuc. Acid. Res. 17:147-161, (1990) New Biol. 2:441-449). Crerecombinase is a 34 kDa protein that catalyzes recombination between twoof its recognition sites called loxP. The loxP site is a 34 base pairconsensus sequence consisting of a core spacer sequence of 8 base pairsand two flanking 13 base pair palindromic sequences.

One of the key advantages to this system is that there is no need foradditional co-factors or sequence elements for efficient recombinationregardless of cellular environment. Recombination occurs within thespacer area of the loxP sites. The post-recombination loxP sites areformed from the two complementary halves of the pre-recombination sites.The result of the Cre recombinase-mediated recombination depends on thelocation and orientation of the loxP sites. When an intervening sequenceis flanked by similarly oriented loxP sites, as in the presentinvention, Cre recombinase activity results in excision. Cre/loxPrecombination can be used at a high efficiency to excise a transgene invivo (Orban et al. (1992) Proc. Natl. Acad. Sci. USA 89: 6861-6865).

Sleeping Beauty Transposon

In another embodiment, the transgenic construct incorporates theinverted repeats of the Sleeping Beauty (SB) transposon system such thatactivation of transposition by SB10 transposase integrates the gene ofinterest and/or the SRC; optionally flanked by loxp recombination sites,into various regions of the genome (Izsvak et al., J. Mol. Biol. (2000)302:93-102).

The SB transposition system is a “cut-and-paste” type transposon memberof the Tc1/mariner superfamily of salmonid transposable elements and isthe only active DNA-based transposon system of vertebrate origincurrently available for experimental manipulation.

Tc1/mariner transposons and transformation systems comprising them aredescribed in U.S. Pat. Nos. 6,225,121, 6,159,717, 5,840,865, 5,348,874to Savakis et al., and U.S. Pat. No. 6,051,430 to Plasterk et al., allof which are incorporated by reference in their entirety.

SB transposon functions in salmon, carp, mouse, and human cells.(Izsvak, supra.). Horie et al., Proc. Natl. Acad. Sci. USA. (2001) 98:9191-9196, describes SB transposon-mediated creation of transgenic mouselines expressing Enhanced Green Fluorescent Protein). There are twofundamental components of any mobile cut-and-paste transposition system:the first is an active transposase and the second is a DNA sequencerecognized and mobilized by the transposase (Ivics et al., supra). SBhas an inverted repeat/direct repeat (IR/DR) structure: directlyrepeated DNA sequence motifs at the ends of each approximately 230 bpimperfect inverted repeats. (Matches are less than 80% at the center ofIRs; Ivics et al., supra.) The direct repeats (perfect repeats) are thecore components of the binding sites for SB transposase. SBtransposition requires the presence of two transposase binding siteswithin each inverted repeat. Mutant transposons having either insertionsor deletions of any or all of any one binding site demonstrate asignificant reduction in transposition efficiency. One mutant transposonthat has one intact inverted repeat (having both transposase bindingsites) and one partially deleted inverted repeat sequence (having onlyone functioning transposase binding site) demonstrated 26% transpositionefficiency (Izsvak et al., supra). Therefore, mutants with partialfunction (defined herein as “partially functional mutants”) of SB arewithin the scope of this invention and preferably have at least about20% of “normal” SB function. This permits a degree of control overtransposition frequency and number of transposed DNA copies intransfectants.

One presently available SB transposase and encoding nucleic acid (SB10transposase) is a synthetic construct bringing together known functionaldomains from public domain nucleic (Tss1.1 element from Atlantic Salmon,GenBank accession #L12206; and Tss1.2 element from Atlantic Salmon,accession #L12207, (Ivics et al., supra). This construct is available asplasmid, pCMV-SB. The nucleic acids including wild type and mutant SBinverted repeats are also available as plasmid, pT/MCS.

In one embodiment of the invention utilizing this transposition system,the germ cells of sterile males deficient in spermatogenesis areco-transfected with both a transgenic construct flanked by nucleic acidsencoding the SB transposon inverted repeats (FIG. 3) and a plasmidcomprising a DNA encoding the SB transposase (preferably pCMV-SB). SBtransposase expression following transfection results in transpositionof the transgenic construct and incorporation into multiple integrationsites in the genome (FIG. 4).

In another embodiment employing this system, SB transposase encoding DNAwith an operatively linked promoter are incorporated into thetransfected transgenic construct.

Genetic Crosses

The general breeding scheme employed to carry out genetic crosses thatproduce transgenic progeny is illustrated in FIG. 1.

After electroporation or other form of DNA uptake, the presumptivelytransgenic mouse is allowed to recover from the surgery and to mature.When sexually mature, it is mated to a female mouse. If the Cre/loxPsystem is used, the female is transgenic for Cre recombinase encodingnucleic acid under the control of hCMV promoter.

If the SB transposition system is used, it can be activated either byco-transfection; as described above, or by mating the male transfectedwith the transgenic construct with a female that is transgenic for theSB transposase which mediates transposition of the region flanked by theSB inverted repeat into other locations of the transgenic mouse genome.

The F₁ progeny of the above types of crosses are genotyped to ensurethat they have the relevant transgenes in their respective genomes.

The SEFs will be excised from the germline in the F₂ generation at thetime that Cre recombinase acts at the loxP recombination sites ofprogeny of the F₁ mating.

The hCMV-Cre transgene segregates out by crossing these F₁ progeny to awild type strain to yield an F₂ generation having only the transgenederived nucleic acid of interest flanked by a single upstream loxP site.

The references cited above are all incorporated by reference herein,whether specifically incorporated or not.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

1. A nucleic acid construct for introducing a transgene into a mammalcomprising (a) a nucleic acid of interest which is optionallyoperatively linked to a first promoter, (b) a spermatogenesis rescuecassette comprising a nucleic acid encoding a spermatogenesis essentialfactor (SEF) operatively linked to a second promoter.
 2. The constructof claim 1 wherein the spermatogenesis rescue cassette is flanked at its5′ and 3′ end by a loxP recombination site.
 3. he construct of claim 1wherein the SEF is selected from the group consisting of Dmc1, Prp8 andCcna1. 4-5. canceled
 6. The construct of claim 1 wherein the firstpromoter is an hCMV promoter or an hEF promoter and the second promoteris (a) an hCMV promoter (b) an hEF promoter; (c) the H1t promoter or (d)a promoter natively linked to a SEF gene encoding said SEF. 7-8.canceled
 9. The construct of claim 1 further comprising at its 5′ and 3′ends, (i) nucleic acid sequences encoding SB transposon inverted repeatsor (ii) a partially functional mutant of said repeat-encoding sequence.10. The construct of claim 6 further comprising at its 5′ and 3′ ends,(i) nucleic acid sequences encoding SB transposon inverted repeats or(ii) a partially functional mutant of said repeat-encoding sequence.11-12. canceled
 13. The construct of claim 9 further comprising anucleic acid sequence encoding a functional transposase that recognizessaid inverted repeats and a promoter operatively linked thereto.
 14. Theconstruct of claim 10 further comprising a nucleic acid sequenceencoding a functional transposase that recognizes said inverted repeatsand a promoter operatively linked thereto. 15-16. canceled
 17. A methodof producing a transgenic mouse that expresses a nucleic acid ofinterest, comprising the steps of: (a) transfecting germ cells of asterile spermatogenesis-deficient immature male mouse with the constructof claim 1 which incorporates both said spermatogenesis rescue cassetteand said nucleic acid of interest into said germ cells, therebygenerating a transfectant mouse; (b) permitting expression of said SEFin said transfectant mouse, thereby rescuing spermatogenesis; (c)waiting a sufficient period for said transfectant mouse to maturesexually; (d) mating the transfectant mouse to a female mouse to createF₁ progeny mice; and (e) mating a resulting F₁ progeny mouse to afertile mouse, thereby producing said transgenic mouse whose genomeincludes said nucleic acid of interest.
 18. A method of producing atransgenic mouse that expresses a nucleic acid of interest, comprisingthe steps of: (a) transfecting germ cells of a sterilespermatogenesis-deficient immature male mouse with the construct ofclaim 6 which incorporates both said spermatogenesis rescue cassette andsaid nucleic acid of interest into said germ cells, thereby generating atransfectant mouse; (b) permitting expression of said SEF in saidtransfectant mouse, thereby rescuing spermatogenesis; (c) waiting asufficient period for said transfectant mouse to mature sexually; (d)mating the transfectant mouse to a female mouse to create F₁ progenymice; and (e) mating a resulting F₁ progeny mouse to a fertile mouse,thereby producing said transgenic mouse whose genome includes saidnucleic acid of interest. 19-20. canceled
 21. A method of producing atransgenic mouse that expresses a nucleic acid of interest, comprisingthe steps of: (a) transfecting germ cells of a sterilespermatogenesis-deficient immature male mouse with the construct ofclaim 9 which incorporates both said spermatogenesis rescue cassette andsaid nucleic acid of interest into said germ cells, thereby generating atransfectant mouse; (b) permitting expression of said SEF in saidtransfectant mouse, thereby rescuing spermatogenesis; (c) waiting asufficient period for said transfectant mouse to mature sexually; (d)mating the transfectant mouse to a female mouse to create F₁ progenymice; and (e) mating a resulting F₁ progeny mouse to a fertile mouse,thereby producing said transgenic mouse whose genome includes saidnucleic acid of interest.
 22. A method of producing a transgenic mousecomprising: (a) transfecting germ cells of a sterilespermatogenesis-deficient male mouse with the construct of claim 1 whichincorporates both said spermatogenesis rescue cassette and said nucleicacid of interest into said germ cells, thereby generating a transfectantmouse; (b) permitting expression of said SEF in said transfectant mouse,thereby rescuing spermatogenesis; (c) waiting a sufficient period forsaid transfectant mouse to mature sexually; (d) mating the transfectantmouse to a female mouse that is transgenic for DNA encoding Crerecombinase to create F₁ progeny mice, wherein, when said constructincludes said loxP sites, said Cre recombinase mediates excision of saidspermatogenesis rescue cassette from the DNA of said F₁ progeny mice;and (e) mating a resulting F₁ progeny mouse to a fertile mouse, therebyproducing said transgenic mouse whose genome includes said transgenicnucleic acid of interest but not said spermatogenesis rescue cassette.23. A method of producing a transgenic mouse comprising: (a)transfecting germ cells of a sterile spermatogenesis-deficient malemouse with the construct of claim 6 which incorporates both saidspermatogenesis rescue cassette and said nucleic acid of interest intosaid germ cells, thereby generating a transfectant mouse; (b) permittingexpression of said SEF in said transfectant mouse, thereby rescuingspermatogenesis; (c) waiting a sufficient period for said transfectantmouse to mature sexually; (d) mating the transfectant mouse to a femalemouse that is transgenic for DNA encoding Cre recombinase to create F₁progeny mice, wherein, when said construct includes said loxP sites,said Cre recombinase mediates excision of said spermatogenesis rescuecassette from the DNA of said F₁ progeny mice; and (e) mating aresulting F₁ progeny mouse to a fertile mouse, thereby producing saidtransgenic mouse whose genome includes said transgenic nucleic acid ofinterest but not said spermatogenesis rescue cassette. 24-25. canceled26. A method of producing a transgenic mouse comprising: (a)transfecting germ cells of a sterile spermatogenesis-deficient malemouse with the construct of any of claim 9 which incorporates both saidspermatogenesis rescue cassette and said nucleic acid of interest intosaid germ cells, thereby generating a transfectant mouse; (b) permittingexpression of said SEF in said transfectant mouse, thereby rescuingspermatogenesis; (c) waiting a sufficient period for said transfectantmouse to mature sexually; (d) mating the transfectant mouse to a femalemouse that is transgenic for DNA encoding Cre recombinase to create F₁progeny mice, wherein, when said construct includes said loxP sites,said Cre recombinase mediates excision of said spermatogenesis rescuecassette from the DNA of said F₁ progeny mice; and (e) mating aresulting F₁ progeny mouse to a fertile mouse, thereby producing saidtransgenic mouse whose genome includes said transgenic nucleic acid ofinterest but not said spermatogenesis rescue cassette.
 27. The method ofclaim 17 wherein (i) said construct includes said loxP sites, (ii) saidfemale mouse of step (d) is transgenic for DNA encoding Cre recombinase,so that in said F₁ progeny mice, said recombinase mediates excision ofsaid spermatogenesis rescue cassette from the DNA, thereby producing atransgenic mouse whose genome includes said transgenic nucleic acid ofinterest but not said spermatogenesis rescue cassette.
 28. The method ofclaim 18 wherein (i) said construct includes said loxP sites, (ii) saidfemale mouse of step (d) is transgenic for DNA encoding Cre recombinase,so that in said F₁ progeny mice, said recombinase mediates excision ofsaid spermatogenesis rescue cassette from the DNA, thereby producing atransgenic mouse whose genome includes said transgenic nucleic acid ofinterest but not said spermatogenesis rescue cassette. 29-30. canceled31. The method of claim 21 wherein (i) said construct includes said loxPsites, (ii) said female mouse of step (d) is transgenic for DNA encodingCre recombinase, so that in said F₁ progeny mice, said recombinasemediates excision of said spermatogenesis rescue cassette from the DNA,thereby producing a transgenic mouse whose genome includes saidtransgenic nucleic acid of interest but not said spermatogenesis rescuecassette.
 32. The method of claim 22 wherein (i) said construct includessaid loxP sites, (ii) said female mouse of step (d) is transgenic forDNA encoding Cre recombinase, so that in said F₁ progeny mice, saidrecombinase mediates excision of said spermatogenesis rescue cassettefrom the DNA, thereby producing a transgenic mouse whose genome includessaid transgenic nucleic acid of interest but not said spermatogenesisrescue cassette.
 33. The method of claim 23 wherein (i) said constructincludes said loxP sites, (ii) said female mouse of step (d) istransgenic for DNA encoding Cre recombinase, so that in said F₁ progenymice, said recombinase mediates excision of said spermatogenesis rescuecassette from the DNA, thereby producing a transgenic mouse whose genomeincludes said transgenic nucleic acid of interest but not saidspermatogenesis rescue cassette. 34-35. canceled
 36. The method of claim26 wherein (i) said construct includes said loxP sites, (ii) said femalemouse of step (d) is transgenic for DNA encoding Cre recombinase, sothat in said F₁ progeny mice, said recombinase mediates excision of saidspermatogenesis rescue cassette from the DNA, thereby producing atransgenic mouse whose genome includes said transgenic nucleic acid ofinterest but not said spermatogenesis rescue cassette.
 37. The method ofclaim 17 wherein the transfecting is by electroporation, nucleic acidcoated microprojectile bombardment or lipid mediated transfection.38-39. canceled
 40. The method of claim 22 wherein the transfecting isby electroporation, nucleic acid coated microprojectile bombardment orlipid mediated transfection. 41-45. canceled
 46. The method of claim 32wherein the transfecting is by electroporation, nucleic acid coatedmicroprojectile bombardment or lipid mediated transfection. 47-48.canceled
 49. The method of claim 17 wherein the transfecting stepfurther comprises co-transfecting said germ cells of said sterilespermatogenesis-deficient male mouse with a plasmid comprising DNAencoding an SB transposase, which method further comprises, aftertransfection, the step of expressing said SB transposase such thattransposon mediated integration incorporates the construct in one ormore sites of the transfected mouse genome. 50-53. canceled
 54. Themethod of claim 22 wherein the transfecting step further comprisesco-transfecting said germ cells of said sterilespermatogenesis-deficient male mouse with a plasmid comprising DNAencoding an SB transposase, which method further comprises, aftertransfection, the step of expressing said SB transposase such thattransposon mediated integration incorporates the construct in one ormore sites of the transfected mouse genome. 55-69. canceled
 70. Themethod of claim 17 wherein the construct used in the transfecting stepfurther comprises DNA encoding an SB transposase and an operativelylinked promoter, said method further comprising, after transfection, thestep of expressing said SB transposase such that transposon mediatedintegration incorporates the construct in one or more sites of thetransfected mouse genome.
 71. The method of claim 22 wherein theconstruct used in the transfecting step further comprises DNA encodingan SB transposase and an operatively linked promoter, said methodfurther comprising, after transfection, the step of expressing said SBtransposase such that transposon mediated integration incorporates theconstruct in one or more sites of the transfected mouse genome. 72.canceled
 73. The method of claim 32 wherein the construct used in thetransfecting step further comprises DNA encoding an SB transposase andan operatively linked promoter, said method further comprising, aftertransfection, the step of expressing said SB transposase such thattransposon mediated integration incorporates the construct in one ormore sites of the transfected mouse genome.
 74. The method of claim 17wherein the female mouse of the mating step is further transgenic forDNA encoding SB transposase, and further comprising after mating thestep of expressing said SB transposase such that transposon mediatedintegration incorporates the construct in one or more sites of thetransfected mouse genome.
 75. The method of claim 22 wherein the femalemouse of the mating step is further transgenic for DNA encoding SBtransposase, and further comprising after mating the step of expressingsaid SB transposase such that transposon mediated integrationincorporates the construct in one or more sites of the transfected mousegenome.
 76. The method of claim 27 wherein the female mouse of themating step is further transgenic for DNA encoding SB transposase, andfurther comprising after mating the step of expressing said SBtransposase such that transposon mediated integration incorporates theconstruct in one or more sites of the transfected mouse genome.
 77. Themethod of claim 32 wherein the female mouse of the mating step isfurther transgenic for DNA encoding SB transposase, and furthercomprising after mating the step of expressing said SB transposase suchthat transposon mediated integration incorporates the construct in oneor more sites of the transfected mouse genome.
 78. A transgenic mousewhose germline cells comprise a transgene which includes: (i) a nucleicacid of interest, operatively linked to a first promoter; andexpressible in cells of said transgenic mouse; and (ii) aspermatogenesis rescue cassette comprising a nucleic acid encoding a SEFoperatively linked to a second promoter, which cassette is optionallyflanked at its 5′ and 3′ end by a loxP recombination site.
 79. Atransgenic mouse whose cells comprise: (i) a transgenic nucleic acid ofinterest, operatively linked to a first promoter; and expressible incells of said transgenic mouse; and (ii) a single loxP recombinationsite either upstream or downstream from said nucleic acid of interest.80. The transgenic mouse of claim 78 wherein the SEF is Dmc1, Prp orCcna1. 81-82. canceled
 83. The transgenic mouse of claim 78 wherein thefirst promoter is an hCMV promoter or a hEF promoter and the secondpromoter is the H1t promoter or a promoter natively linked to the SEFgene. 84-88. canceled
 89. The transgenic mouse of claim 79 wherein thefirst promoter is an hCMV promoter or a hEF promoter and the secondpromoter is the H1t promoter or a promoter natively linked to the SEFgene. 90-93. canceled
 94. The transgenic mouse of claim 78 furthercomprising (i) nucleic acid sequences encoding SB transposon invertedrepeats or (ii) a partially functional mutant of said repeat-encodingsequence.
 95. The transgenic mouse of claim 79 further comprising (i)nucleic acid sequences encoding SB transposon inverted repeats or (ii) apartially functional mutant of said repeat-encoding sequence.
 96. Thetransgenic mouse of claim 80 further comprising (i) nucleic acidsequences encoding SB transposon inverted repeats or (ii) a partiallyfunctional mutant of said repeat-encoding sequence. 97-98. canceled 99.The transgenic mouse of claim 83 further comprising (i) nucleic acidsequences encoding SB transposon inverted repeats or (ii) a partiallyfunctional mutant of said repeat-encoding sequence.